CN104792771A - Method for detecting HRP through luminol-hydrogen peroxide-bromophenol red-HRP-BSA chemiluminescent system - Google Patents
Method for detecting HRP through luminol-hydrogen peroxide-bromophenol red-HRP-BSA chemiluminescent system Download PDFInfo
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Abstract
The invention relates to a method for detecting HRP through luminol-hydrogen peroxide-bromophenol red-HRP-BSA chemiluminescent system and aims to build a novel chemiluminescent system which is used for high sensitivity detection of HRP. The chemiluminescent system comprises liquid A, liquid B and liquid C, wherein the liquid A is mixed liquid of bromophenol red and hydrogen peroxide; the liquid B is luminol; the liquid C is a BSA aqueous solution; a sample HRP to be detected is diluted through the liquid C; the diluted sample is placed in a measuring cup and the liquid A is added for light shielding reaction; the liquid B is added and chemiluminescence signal strength determination is carried out in a weak chemiluminescent analyzer. The method has the advantages of higher sensitivity and better linearity on the aspect of HRP detection in comparison with a method adopting a classic luminol-hydrogen peroxide-HRP-PIP chemiluminescent system.
Description
Technical field:
The present invention relates to the detection method of horseradish peroxidase, relate to the method that a kind of luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system detects HRP further.
Background technology:
Chemiluminescence (Chemiluminescence, CL) analyzing is a kind of trace analysis method producing optical radiation determination content of material according to chemical reaction, because it is highly sensitive, the range of linearity is wide, instrument and equipment is simple, easy to operate, analyze and realize robotization fast and easily, to become in analytical chemistry a very active study hotspot in recent years, and intersect with numerous subject, investigation and application field is more and more extensive.
Luminol-hydrogen peroxide-horseradish peroxidase (Horseradish Peroxidase, HRP) chemical luminous system is a kind of conventional chemiluminescence analysis system.Due to HRP can with different kinds of molecules coupling, the conventional HRP (Biotin-HRP) including biotin modification, the HRP (SA-HRP) that Streptavidin is modified, the HRP etc. that second antibody is modified, HRP can also be marked on various Stationary liquid (polystyrene microsphere and magnetic bead etc. as golden nanometer particle, carboxyl modified), therefore Western blotting is widely used in, the detection of Immunohistochemical Method and chemiluminescent enzyme immunoassay (CLEIA) middle DNA, antibody and tumor marker etc.Chemiluminescence intensity due to luminol-hydrogen peroxide-HRP chemical luminous system is not high and easily decay, and therefore this system needs to add reinforcing agent usually.It is the classical reinforcing agent of luminol-hydrogen peroxide-HRP system to iodophenol (4-Iodophenol, PIP).
Summary of the invention:
The object of the invention is to set up novel luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system, use it for high-sensitivity detection HRP.
In the said novel luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system of this patent, luminol is chemiluminescence agent, and hydrogen peroxide is oxygenant, and HRP is testing sample, and BSA and dibromophenolphthalein are associating reinforcing agents.
Having the present invention relates to a kind of novel reinforcing agent is dibromophenolphthalein, and Chinese another name dibromophenol sulfonephthalein is a kind of phenol derivatives.
Also known as the 5th component, molecular weight is about 67kDa to bovine serum albumin(BSA) (Bovine Serum Albumin, BSA), and it contains 607 amino acid residues sorted successively, and constitutes 3 columniform hydrophobic cavities at intramolecule.BSA is the protein that in serum, content is the highest, and to maintaining the osmotic equilibrium of blood, the transhipment of exogenous and endogenic ligand, distribution, metabolism all play an important role.It is that medicine has the main associated proteins of physiological action compound with other.Because BSA is cheap and easy to get, stable in properties, has very strong binding ability to medicine, and therefore it is widely used in the research in Biochemical Research, genetic engineering and medical research field.Find that BSA and New Dry Strength Agents dibromophenolphthalein have the luminol-luminous signal of hydrogen peroxide-HRP chemiluminescence body of light system herein and combine humidification significantly, based on this, luminol-hydrogen peroxide that the present invention adopts this novel-dibromophenolphthalein-BSA chemical luminous system, high-sensitivity detection HRP.
Described in this patent, novel light-emitting system comprises A liquid, B liquid and C liquid; A liquid is dibromophenolphthalein, 0.1-10mM, 0.2M HAc-KAc damping fluid, pH 5.25) with hydrogen peroxide (0.1-5mM, the water) mixed liquor that mixes of 1:4 by volume before use; B liquid is luminol (0.01-5mM, 0.1M Tris damping fluid, pH 11.0); C liquid is the BSA aqueous solution of 0.001%-1%.During measurement, get testing sample HRP 10 μ L and be placed in 14 × 40mm measuring cup, add A liquid portion, lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carries out chemiluminescence signal strength detection.
The present invention is relative to prior art, and tool has the following advantages:
Dibromophenolphthalein is the reinforcing agent of a kind of novel luminol-hydrogen peroxide-HRP chemical luminous system, by many experiments, inventor finds that luminol-hydrogen peroxide-HRP chemical luminous system of strengthening dibromophenolphthalein of BSA has and significantly combines humidification, can significantly improve the detection sensitivity of HRP.
Carry out comparative study with classical luminol-hydrogen peroxide-HRP-PIP chemical luminous system, find that the present invention has higher sensitivity with better linear in detection HRP.
Accompanying drawing explanation
Fig. 1-16 is embodiment test result figure, and in figure, horizontal ordinate represents HRP concentration, ng mL
-1; Ordinate represents chemiluminescence signal strength values.
Fig. 1 is embodiment 1 test result figure; When C liquid is 0.001%BSA aqueous solution, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ngmL
-1, the range of linearity is 1-6ng mL
-1, when HRP concentration is 5ng mL
-1time, the recovery is 88% ± 6%.
Fig. 2 is embodiment 2 test result figure; When C liquid is 0.01%BSA aqueous solution, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 1ngmL
-1, the range of linearity is 1.5-7ng mL
-1, when HRP concentration is 5ng mL
-1time, the recovery is 103% ± 3%.
Fig. 3 is embodiment 3 test result figure; When C liquid is 0.1%BSA aqueous solution, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ng mL
-1, the range of linearity is 1-5ng mL
-1, when HRP concentration is 2ng mL
-1time, the recovery is 105% ± 3%.
Fig. 4 is embodiment 4 test result figure; When C liquid is 0.5%BSA aqueous solution, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ng mL
-1, the range of linearity is 1-5ng mL
-1, when HRP concentration is 4ng mL
-1time, the recovery is 103% ± 3%.
Fig. 5 is embodiment 5 test result figure; When C liquid is 1%BSA aqueous solution, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ngmL
-1, the range of linearity is 1.5-9ng mL
-1, when HRP concentration is 2.5ng mL
-1time, the recovery is 92% ± 7%.
Fig. 6 is embodiment 6 test result figure; When B liquid is 0.01mM luminol, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ngmL
-1, the range of linearity is 1-7ng mL
-1, when HRP concentration is 3ng mL
-1time, the recovery is 106% ± 5%.
Fig. 7 is embodiment 7 test result figure; When B liquid is 0.1mM luminol, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ngmL
-1, the range of linearity is 1-7ng mL
-1, when HRP concentration is 1.5ng mL
-1time, the recovery is 109% ± 3%.
Fig. 8 is embodiment 8 test result figure; When B liquid is 0.5mM luminol, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ngmL
-1, the range of linearity is 1-9ng mL
-1, when HRP concentration is 3ng mL
-1time, the recovery is 105% ± 3%.
Fig. 9 is embodiment 9 test result figure; When B liquid is 5mM luminol, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ngmL
-1, the range of linearity is 1-7ng mL
-1, when HRP concentration is 2ng mL
-1time, the recovery is 93% ± 4%.
Figure 10 is embodiment 10 test result figure; When A liquid be 0.1mM dibromophenolphthalein and 1mM hydrogen peroxide 1:4 mixes by volume time, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ng mL
-1, the range of linearity is 1-6ng mL
-1, when HRP concentration is 2ng mL
-1time, the recovery is 105% ± 5%.
Figure 11 is embodiment 11 test result figure; When A liquid be 1mM dibromophenolphthalein and 1mM hydrogen peroxide 1:4 mixes by volume time, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ng mL
-1, the range of linearity is 0.5-7ng mL
-1, when HRP concentration is 2ng mL
-1time, the recovery is 109% ± 3%.
Figure 12 is embodiment 12 test result figure; When A liquid be 10mM dibromophenolphthalein and 1mM hydrogen peroxide 1:4 mixes by volume time, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ng mL
-1, the range of linearity is 1-6ng mL
-1, when HRP concentration is 3ng mL
-1time, the recovery is 101% ± 3%.
Figure 13 is embodiment 13 test result figure; When A liquid be 4mM dibromophenolphthalein and 0.1mM hydrogen peroxide 1:4 mixes by volume time, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ng mL
-1, the range of linearity is 0.5-5ng mL
-1, when HRP concentration is 1.5ng mL
-1time, the recovery is 105% ± 3%.
Figure 14 is embodiment 14 test result figure; When A liquid be 4mM dibromophenolphthalein and 0.5mM hydrogen peroxide 1:4 mixes by volume time, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 0.5ng mL
-1, the range of linearity is 0.5-9ng mL
-1, when HRP concentration is 2ng mL
-1time, the recovery is 113% ± 6%.
Figure 15 is embodiment 15 test result figure; When A liquid be 4mM dibromophenolphthalein and 2.5mM hydrogen peroxide 1:4 mixes by volume time, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 1ng mL
-1, the range of linearity is 2-10ng mL
-1, when HRP concentration is 4ng mL
-1time, the recovery is 93% ± 3%.
Figure 16 is embodiment 16 test result figure; When A liquid be 4mM dibromophenolphthalein and 5mM hydrogen peroxide 1:4 mixes by volume time, this new chemical luminescence system detects the typical curve of HRP, and its detection is limited to 2ng mL
-1, the range of linearity is 3-9ng mL
-1, when HRP concentration is 5ng mL
-1time, the recovery is 89% ± 3%.
Figure 17 is embodiment 17 test result figure; In figure, horizontal ordinate represents SA-HRP concentration, unit ng mL
-1; Ordinate represents chemiluminescence signal strength values.When C liquid is 0.1%BSA aqueous solution, this new chemical luminescence system detects the typical curve of SA-HRP, and its detection is limited to 0.1ng mL
-1, the range of linearity is 0.25-6ng mL
-1, when SA-HRP concentration is 5ng mL
-1time, the recovery is 78% ± 3%.
Figure 18 is embodiment 18 test result figure; In figure, horizontal ordinate represents Biotin-HRP concentration, unit ng mL
-1; Ordinate represents chemiluminescence signal strength values.When C liquid is 0.1%BSA aqueous solution, this new chemical luminescence system detects the typical curve of Biotin-HRP, and its detection is limited to 0.5ng mL
-1, the range of linearity is 1-6ng mL
-1, when Biotin-HRP concentration is 1.5ng mL
-1time, the recovery is 114% ± 3%.
Figure 19 is embodiment 19 test result figure; In figure, horizontal ordinate represents SA-HRP concentration, unit ng mL
-1; Ordinate represents chemiluminescence signal strength values.When C liquid is 0.5%BSA aqueous solution, this new chemical luminescence system detects the typical curve of SA-HRP, and its detection is limited to 0.1ng mL
-1, the range of linearity is 0.5-4ng mL
-1, when SA-HRP concentration is 3ng mL
-1time, the recovery is 101% ± 3%.
Figure 20 is embodiment 20 test result figure; In figure, horizontal ordinate represents Biotin-HRP concentration, unit ng mL
-1; Ordinate represents chemiluminescence signal strength values.When C liquid is 0.5%BSA aqueous solution, this new chemical luminescence system detects the typical curve of Biotin-HRP, and its detection is limited to 0.5ng mL
-1, the range of linearity is 1.5-6ng mL
-1, when Biotin-HRP concentration is 2ng mL
-1time, the recovery is 102% ± 3%.
Figure 21-23 is test result figure of comparative example 1-3;
Figure 21 is the test result figure of comparative example 1; In figure, horizontal ordinate represents HRP concentration, unit ng mL
-1; Ordinate represents chemiluminescence signal strength values.
Figure 22 is the test result figure of comparative example 2; In figure, horizontal ordinate represents Biotin-HRP concentration, unit ng mL
-1; Ordinate represents chemiluminescence signal strength values.
The test result figure of Figure 23 comparative example 3; In figure, horizontal ordinate represents SA-HRP concentration, unit ng mL
-1; Ordinate represents chemiluminescence signal strength values.
Embodiment
Experimental procedure:
By dibromophenolphthalein (dilution of 0.1-10mM, pH 5.250.2M HAc-KAc damping fluid) and hydrogen peroxide (0.1-5mM, water dilutes) by volume 1:4 mix before use, as A liquid; Luminol (dilution of 0.01-5mM, pH 11.00.1M Tris damping fluid) is as B liquid; 0.001%-1%BSA (water dilution) is C liquid; Testing sample HRP C liquid dilutes.Testing sample HRP 10 μ L is placed in 14 × 40mm measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.
In following examples, NM condition is all identical with the condition in above-mentioned steps.
Embodiment 1: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.001%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 8 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,3,4,6,7ng mL
-1.Experimental result as shown in Figure 1.
Embodiment 2: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.01%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 9 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,2,4,6,7,9ng mL
-1.Experimental result as shown in Figure 2.
Embodiment 3: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 9 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,3,4,5,6,7ng mL
-1.Experimental result as shown in Figure 3.
Embodiment 4: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.5%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 7 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,2,3,5ng mL
-1.Experimental result as shown in Figure 4.
Embodiment 5: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 10 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,2,3,4,6,7,9ng mL
-1.Experimental result as shown in Figure 5.
Embodiment 6: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 0.01mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 8 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,2,4,7,9ng mL
-1.Experimental result as shown in Figure 6.
Embodiment 7: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 0.1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 9 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,2,5,6,7,9,10ng mL
-1.Experimental result as shown in Figure 7.
Embodiment 8: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 0.5mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 8 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,2,4,9,10ng mL
-1.Experimental result as shown in Figure 8.
Embodiment 9: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 5mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 9 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,3,4,5,6,7ng mL
-1.Experimental result as shown in Figure 9.
Embodiment 10: by 0.1mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 9 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,3,4,5,6,7ng mL
-1.Experimental result as shown in Figure 10.
Embodiment 11: by 1mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 10 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,3,5,6,7,9,10ng mL
-1.Experimental result as shown in figure 11.
Embodiment 12:: by 10mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 8 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,2,4,6,7ng mL
-1.Experimental result as shown in figure 12.
Embodiment 13: by 4mM dibromophenolphthalein and 0.1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 8 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,2,4,5,6,7ng mL
-1.Experimental result as shown in figure 13.
Embodiment 14: by 4mM dibromophenolphthalein and 0.5mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 9 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.5,1,1.5,3,4,7,9,10ng mL
-1.Experimental result as shown in figure 14.
Embodiment 15: by 4mM dibromophenolphthalein and 2.5mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 9 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,0.1,2,3,5,7,9,10,11ng mL
-1.Experimental result as shown in figure 15.
Embodiment 16: by 4mM dibromophenolphthalein and 5mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample HRP C liquid dilutes.Getting 10 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample HRP concentration is respectively 0,1,1.5,2,3,4,6,7,9,10ng mL
-1.Experimental result as shown in figure 16.
Embodiment 17: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample SA-HRP C liquid dilutes.Getting 8 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample SA-HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample SA-HRP concentration is respectively 0,0.1,0.25,0.5,2,4,6,7ng mL
-1.Experimental result as shown in figure 17.
Embodiment 18: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.1%BSA is C liquid; Testing sample Biotin-HRP C liquid dilutes.Getting 8 specifications is the measuring cup of 14 × 40mm, add finite concentration testing sample Biotin-HRP 10 μ L in each measuring cup, add A liquid portion, lucifuge reaction 30min, add B liquid a, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample Biotin-HRP concentration is respectively 0,0.5,1,2,4,5,6,7ng mL
-1.Experimental result as shown in figure 18.
Embodiment 19: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.5%BSA is C liquid; Testing sample SA-HRP C liquid dilutes.Getting 8 specifications is the measuring cup of 14 × 40mm, adds finite concentration testing sample SA-HRP 10 μ L in each measuring cup, adds A liquid portion, and lucifuge reaction 30min, adds B liquid portion, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample SA-HRP concentration is respectively 0,0.1,0.25,0.5,1,1.5,2,4ng mL
-1.Experimental result as shown in figure 19.
Embodiment 20: by 4mM dibromophenolphthalein and 1mM hydrogen peroxide by volume 1:4 mix before use, as A liquid; 1mM luminol is as B liquid; 0.5%BSA is C liquid; Testing sample Biotin-HRP C liquid dilutes.Getting 8 specifications is the measuring cup of 14 × 40mm, add finite concentration testing sample Biotin-HRP 10 μ L in each measuring cup, add A liquid portion, lucifuge reaction 30min, add B liquid a, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.Testing sample Biotin-HRP concentration is respectively 0,0.5,1,1.5,3,4,5,6,7ng mL
-1.Experimental result as shown in figure 20.
Comparative example 1: getting 7 specifications is the measuring cup of 14 × 40mm, adds 4mM PIP (0.1M Tris damping fluid, pH 8.3), 0.2 part in each measuring cup; Finite concentration testing sample HRP aqueous solution, 10 μ L; 1mM aqueous hydrogen peroxide solution, 0.8 part; 1mM luminol (0.1M Tris damping fluid, pH 11.0) is a, and in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection after mixing, the concentration of testing sample HRP is respectively 0,0.5,0.7,1,1.4,2,5ng mL
-1.Experimental result as shown in figure 21.
Comparative example 2: getting 9 specifications is the measuring cup of 14 × 40mm, adds 4mM PIP (0.1M Tris damping fluid, pH 8.3), 0.2 part in each measuring cup; Finite concentration testing sample Biotin-HRP aqueous solution, 10 μ L; 1mM aqueous hydrogen peroxide solution, 0.8 part; 1mM luminol (0.1M Tris damping fluid, pH 11.0) is a, and in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection after mixing, the concentration of testing sample Biotin-HRP is respectively 0,0.5,1,1.5,2,3,4,5,6ng mL
-1.Experimental result as shown in figure 22.
Comparative example 3: getting 10 specifications is the measuring cup of 14 × 40mm, adds 4mM PIP (0.1M Tris damping fluid, pH 8.3), 0.2 part in each measuring cup; Finite concentration testing sample SA-HRP aqueous solution, 10 μ L; 1mM aqueous hydrogen peroxide solution, 0.8 part; 1mM luminol (0.1M Tris damping fluid, pH 11.0) is a, and in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection after mixing, the concentration of testing sample SA-HRP is respectively 0, and 0.05,0.1,0.25,0.5,1,1.5,2,3,4ng mL
-1.Experimental result as shown in figure 23.
Invention is not to be considered as being limited to instantiation as herein described, and be interpreted as the present invention and cover the of the present invention all aspects intactly listed in the dependent claims.For those skilled in the art in the invention, after reading the present invention, the present invention's various amendment applicatory, equivalent processes and various structure are all apparent.
Claims (10)
1. luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system detects the method for HRP, it is characterized in that:
Luminescence system comprises A liquid, B liquid and C liquid; A liquid is the mixed liquor of dibromophenolphthalein and hydrogen peroxide; B liquid is luminol; C liquid BSA aqueous solution;
Testing sample HRP C liquid dilutes; Be placed in measuring cup, add A liquid, lucifuge is reacted, and adds B liquid, in faint chemiluminescent analyzer, carries out chemiluminescence signal strength detection.
2. luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system detects the method for HRP according to claim 1, it is characterized in that:
A liquid is dibromophenolphthalein and the hydrogen peroxide mixed liquor that mixes of 1:4 by volume before use; Dibromophenolphthalein configuration parameter is: 0.1-10mM, 0.2M HAc-KAc damping fluid, pH 5.25; Hydrogen peroxide configuration parameter is: 0.1-5mM, water.
3. luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system detects the method for HRP according to claim 1, it is characterized in that: B liquid is luminol, and configuration parameter is: 0.01-5mM, 0.1M Tris damping fluid, pH 11.0.
4. luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system detects the method for HRP according to claim 1, it is characterized in that: C liquid is the BSA aqueous solution of 0.001%-1%.
5. detect the method for HRP according to claim 1-5 luminol-hydrogen peroxide described in any one-dibromophenolphthalein-HRP-BSA chemical luminous system, it is characterized in that, testing process is as follows:
Testing sample HRP C liquid dilutes; Be placed in measuring cup, add A liquid, lucifuge reacts 30 minutes; Add B liquid, in faint chemiluminescent analyzer, carry out chemiluminescence signal strength detection.
6. luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system detects the method for HRP according to claim 1, it is characterized in that:
The concentration of dibromophenolphthalein is 4mM, and the concentration of hydrogen peroxide is 1mM; The concentration of luminol is 1mM; The concentration of BSA is 0.1%.
7. luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system detects the method for HRP according to claim 1, it is characterized in that:
The concentration of dibromophenolphthalein is 4mM, and the concentration of hydrogen peroxide is 1mM; The concentration of luminol is 1mM; The concentration of BSA is 0.5%.
8. luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system detects the method for HRP according to claim 1, it is characterized in that:
The concentration of dibromophenolphthalein is 4mM, and the concentration of hydrogen peroxide is 1mM; The concentration of luminol is 0.1mM; The concentration of BSA is 0.1%.
9. luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system detects the method for HRP according to claim 1, it is characterized in that:
The concentration of dibromophenolphthalein is 4mM, and the concentration of hydrogen peroxide is 1mM; The concentration of luminol is 0.5mM; The concentration of BSA is 0.1%.
10. luminol-hydrogen peroxide-dibromophenolphthalein-HRP-BSA chemical luminous system detects the method for HRP according to claim 1, it is characterized in that:
The concentration of dibromophenolphthalein is 4mM, and the concentration of hydrogen peroxide is 2.5mM; The concentration of luminol is 1mM; The concentration of BSA is 0.1%.
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| CN105784660A (en) * | 2016-04-05 | 2016-07-20 | 广西师范学院 | Method for detecting concentration of horseradish peroxidase by utilizing water-soluble InP/ZnS QDs probe |
| CN107024471A (en) * | 2017-04-28 | 2017-08-08 | 福州大学 | A kind of sulfurated hydrogen detection method based on chemical luminous system |
| CN110132944A (en) * | 2019-05-22 | 2019-08-16 | 上海碧云天生物技术有限公司 | Chemical illuminating reagent and its application based on metal ion enhancing |
| CN112326636A (en) * | 2020-10-26 | 2021-02-05 | 通山县金瑞生物科技研发中心 | High-sensitivity ECL kit and preparation method thereof |
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| CN110132944A (en) * | 2019-05-22 | 2019-08-16 | 上海碧云天生物技术有限公司 | Chemical illuminating reagent and its application based on metal ion enhancing |
| CN112326636A (en) * | 2020-10-26 | 2021-02-05 | 通山县金瑞生物科技研发中心 | High-sensitivity ECL kit and preparation method thereof |
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